In genetic engineering, DNA is typically studied by severing long DNA chains into smaller fragments using a restriction enzyme. The resulting fragments, which must then be separated according to size or composition, provide the information needed to construct a map of the original DNA chain. Construction of such a map is facilitated by severing the original DNA chain into a relatively small number of long fragments (preferably less than one hundred), as opposed to generating many short fragments; as the number of pieces decreases, it becomes easier to reconstruct the original molecule. Conventional methods of fragment separation are believed to be limited in that mixtures containing fragments with more than 20,000 base pairs cannot be readily or fully separated. Therefore, with conventional methods of separation, the human chromosomes are cut or severed into thousands of fragments to permit the separation thereof, thus reconstruction of the original chain can be extremely difficult.
In an attempt to alleviate many of these difficulties, variations on the standard known electrophoresis method have been developed. For example, U.S. Pat. No. 4,473,452, hereby incorporated by reference, describes a pulsed field gradient gel electrophoresis method, which in one embodiment involves the application of two nonuniform electric fields positioned at approximately right angles to each other as a means of separating DNA fragments of over 20,000 base pairs. In addition, according to the Abstract of the '452 patent, there is recited an apparatus for and a method of electrophoretically separating particles by electric fields which are transverse to each other, and alternate between respective high and low intensities out of phase with each other at a frequency related to the mass of the particles, thus permitting movement of the particles in an overall direction transverse to the respective directions of the fields. Also, this patent discloses the use of pulsed and crossed gradient electric fields to separate and resolve DNA fragments of up to several million base pairs.
The '452 patent also discloses that particularly good results are obtained when the switching intervals of the alternate fields are proportional to the mass of the particles to be separated raised to a power of about 1.5. More specifically, this patent illustrates that the proper choice of a frequency at which the change from one field to another should occur is related to the time it takes a particle (molecule) of interest to orient itself into an elongated cylindrical shape, and that this time t is related to the mass of the particle (the molecular weight) M, the effective pore radius of the gel r, and the measured velocity of the particle in the gel v, reference column 6, in accordance with the relationship: EQU t.varies.M.sup.1.5 /(r.sup.2 v)
Moreover, in the '452 patent it is indicated that variations such as a differently shaped electrophoresis chamber, or differently produced, distributed or varied electric fields can be used provided that the particles are acted on by electric fields varying with time, permitting them to move in overall directions generally intermediate between at least two of the relevant, operationally significant nonparallel fields. Also, more than two fields can be used providing the net effect is at least to act in the desired manner on a particle first in one direction, then in another direction transverse to the first, thereby moving the particle in a third direction intermediate between the first two. The process of the '452 patent involves the use of crossed alternating inhomogeneous fields to separate large DNA fragments, thereby more complex and costly apparatus is needed, i.e. standard electrophoretic equipment cannot be used.
The electrophoretic process disclosed in the '452 patent is also discussed by C. L. Smith and C. R. Cantor in "Pulsed - Field Gel Electrophoresis of Large DNA Molecules", Nature, Vol. 319, pages 701 to 702 (1986), and by L. M. Corcoran in "Molecular Karyotypes: Separating Chromosomes on Gels", BioAssays, Vol. 3, No. 6, pages 269 to 271 (1985), both of which are hereby incorporated by reference.
Illustrated in U.S. Pat. No. 4,737,251 (WO 87/01955), hereby incorporated by reference, is a method and apparatus for gel electrophoresis employing periodic inversion of the electric field essentially in one dimension, (see Abstract of the Disclosure). Also, according to the Abstract, field inversion gel electrophoresis (FIGE) is used, wherein net migration is achieved by using a longer time, or higher voltage in one direction than in the other direction (see column 2, line 66, to column 3, line 10). Thus, net migration in a given direction can be achieved, for example, by partitioning each switching cycle unequally between the so called forward and reverse directions by imposing a higher voltage in the forward direction than in the reverse direction, and vice versa.
With this process, it is believed that undesirable results, such as the anomalous phenomenon of minimum mobility, are obtained. That is, for example, results where the DNA molecules and the bands they form on an electrophoretic gel during the process do not separate in order of molecular size, preventing quantitative and qualitative analysis of the results because it is not possible to estimate from the results on the gel the size of the molecules separated (see for example FIG. 4 and the description thereof and the working examples, columns 5 to 8).
The '251 patent suggests the use of "switching-interval gradients" (or "ramps") to attempt to reduce the phenomenon of minimum mobility. However, "Ramped Field Inversion Gel Electrophoresis: A Cautionary Note", by T. H. N. Ellis, W. G. Cleary, K. W. G. Burcham and B. A. Bowen, Nucleic Acids Research, Vol. 15, Number 13, 1987, page 5489, teaches that the selection of switching-interval gradients does not eliminate the minimum mobility problem for all molecular sizes, such as large molecular sizes, for example, DNA molecules larger than 1,000 kilobase pairs. Also, the aforesaid problem does not allow one to control systematically the process in such a manner that the final position of all the DNA molecules follows a given pattern in the gel.
The '251 patent discloses a method for the separation of DNA fragments containing 15,000 to over 700,000 base pairs by periodically inverting a uniform electric field of a given strength in one dimension. In one embodiment, this process utilizes fields of equal intensities in both directions with the longest pulse duration in the forward direction. In another embodiment, the use of fields of different intensities with equal pulse durations, the forward field being of larger intensity than the reverse field is suggested. Switching-interval gradients are also suggested in the '251 patent. However, this patent does not teach a process to optimize the separation and minimize, or eliminate the phenomenon of minimum mobility. Also, this patent does not suggest how theoretical and experimental results can be selected and used to design systematic separation strategies where one primary objective is to separate the DNA molecules in such a manner to obtain a predetermined band pattern in the gel after electrophoresis, specifically, for example, a linear or logarithmic pattern with a combination of different pulse intensities and/or durations. Furthermore, this patent indicates that switching-interval gradients can be selected to reduce the minimum mobility problem.
In an article by R. G. Snell and R. J. Wilkins entitled "Separation of Chromosomal DNA Molecules from C. albicans by Pulsed Field Gel Electrophoresis", Nucleic Acids Research, Vol. 14, No. 11, pages 4401 to 4406 (1986), the authors discuss the method of separation apparently disclosed in the '452 patent. The article indicates that variations in experimental conditions such as pulse time, temperature, and relative voltage conditions have critical effects on the quality of results, and that pulsed field gel electrophoresis can be used to resolve DNA from chromosomes of the Candida albicans and Saccharomyces cerevisiae strains of yeast. According to the aforementioned article, the single most important factor for obtaining optimal resolution was the elevation of the electrophoresis temperature to 35.degree. C. Alteration of relative voltage conditions by 10 percent, pulse time by 20 percent, or temperature by 10 percent was, according to this article, found to destroy the electrophoretic pattern.
"Dependence of the Electrophoretic Mobility of DNA in Gels on Field Intermittency", T. Jamil and L. S. Lerman, Journal of Biomolecular Structure and Dynamics, Vol. 2, No. 5, pages 963 to 966 (1985), hereby incorporated by reference, addresses the effect of varying pulse duration and varying the interval between pulses, during which intervals the field is zero depending upon the mobility of DNA fragments in gels. This article illustrates the mobility of lambda DNA fragments containing from 3,400 to 21,800 base pairs when a single pulsed field is applied. The authors concluded that if the interval between pulses remains constant, the apparent mobility increases as the duration of pulses increases, approaching however, a maximum. Additionally, this article discloses that when the pulse duration is constant, the apparent mobility decreases as the interval between pulses becomes longer. This article attributes the changes in apparent mobility due to pulse duration and pulse interval to be relatively small for short fragments of 3,400 base pairs, and quite large for longer fragments of 10,000 base pairs and more. In addition, it is indicated that the dependence of the mobility on pulse interval and duration decreases with decreasing ion concentration in the gel (the authors varied the sodium ion concentration between 0.04 to 0.4M); and these effects become larger with decreasing pore size in agarose. Further, the article presents some mathematical analysis concerning the reasons for the greater effects observed for larger molecules, but provides no quantitative information related to DNA fragments containing more than 22,000 base pairs or to experimental conditions where the field is not zero during the intervals between the main pulses. Also, this article does not appear to mention mathematical analysis as a guide to a process for separating large DNA fragments by choosing optimal experimental conditions for a given mixture of fragments.
In "Prediction of Chain Elongation in the Reptation Theory of DNA Gel Electrophoresis", Biopolymers, Vol. 24, No. 12, pages 2181 to 2184 (1985) and "On the Reptation Theory of Gel Electrophoresis", G. W. Slater and J. Noolandi, Biopolymers, Vol. 25, No. 3, pages 431 to 454 (1986), both of which are hereby incorporated by reference, there is provided a theoretical discussion of the reptation theory of DNA chain motion with respect to gel electrophoresis. These articles disclose three time scales which can be used to calculate optimal experimental conditions for some of the electrophoretic methods that rely on two or more electric field intensities. They do not, however, for example, provide a full quantitative analysis of the correlation between the time scales, the duration and intensities of applied field pulses, and the sizes of DNA fragments to be separated for all experimental systems that use pulsed fields.
Many references disclose the basic process of gel electrophoresis. For example, U.S. Pat. No. 3,630,882, hereby incorporated by reference, teaches an apparatus for particle separation wherein a mixture of particles in a suspending medium is subjected to an intermittent DC electrical field of sufficient strength to produce a sharp separation of two or more components of the mixture. The electric field is intermittent or pulsed so that the particles in the material are alternately subjected to high electric field and low or zero electric field.
Also, U.S. Pat. No. 3,870,612 teaches a method of determining the electrophoretic mobility and diffusion coefficient of a macromolecular polymer in solution wherein the macromolecules are driven through the solution by an electric field in a modified electrophoretic cell. The electric field is pulsed, and the pulses are of alternating polarity to allow for the use of high fields and to prevent formation of concentration gradients.
U.S. Pat. No. 4,148,703, hereby incorporated by reference, illustrates a method of electrophoretic purification of electrically charged biomolecules which uses different geometrically shaped electrode configurations, permitting potentially different gradients and enabling different particle velocities, finer separations, and continuous electrophoresis by means of a higher voltage in a smaller area with a decrease in power expenditure. The various electrode systems are alternately turned on and off at a given time independently of one another and for a given duration of time. Also, in U.S. Pat. No. 3,506,554, there is illustrated a process and apparatus for separating electrophoretically active substances, such as proteins. The method utilizes a continuously flowing stream of buffer to transport the substances through a zone having an inert material that is permeable to either the electrophoretically active material or small buffer ions, such as a polyacrylamide gel slab. The process includes applying an electric field first in one direction and then in another direction to enable separation, and the cycle of reversing the direction of the electric field is repeated many times.
There is disclosed in U.S. Pat. No. 4,061,561 an electrophoretic apparatus that allows for high resolution by performing two dimensional migrations in a square tray. The sample selected is subjected to a linear current in one direction, and the tray is then turned exactly 90.degree. so that the first migration is pulled apart from an orthogonal direction. The '561 patent also discloses a multiple-sample applicator that allows an operator to deposit multiple samples on the gel or membrane either simultaneously or one at a time.
A process and apparatus for purifying and concentrating DNA from a crude DNA - containing mixture, such as whole blood, is disclosed in U.S. Pat. No. 4,617,102, hereby incorporated by reference. The apparatus of the '102 patent consists essentially of an agarose gel disc immersed in an electrophoresis buffer solution and supported between two eight-micrometer polycarbonate filters in an electric field. Placing the sample on the disc and applying an electric field results in the separation of the DNA from the other components of the crude mixture. However, the reference does not appear to teach, for example, a method of separating DNA particles of different molecular weights from each other.
Other documents of interest include U.S. Pat. No. 4,322,275; "Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed - Field Gradient Gel Electrophoresis", K. Gardiner, W. Laas, and D. Patterson, Somatic Cell and Molecular Genetics, Vol. 12, No. 2, pages 185 to 195 (1986); "Mapping of the Class II Region of the Human Major Histocompatibility Complex by Pulsed - Field Gel Electrophoresis", D. A. Hardy et al., Nature, Vol. 323, pages 453 to 455 (1986); "New Biased - Reptation Model for Charged Polymers", G. W. Slater and J. Noolandi, Physical Review Letters, Vol. 55. No. 15, pages 1579 to 1582 (1985); "Scrambling of Bands in Gel Electrophoresis of DNA", M. Lalande, J. Noolandi, C. Turmel, R. Brousseau, J. Rousseau, G. W. Slater, Nucleic Acids Research, Vol. 16, pages 5427 to 5437 (1988); "Pulsed-field Electrophoresis: Application of a Computer Model to the Separation of Large DNA Molecules", M. Lalande, J. Noolandi, C. Turmel, J. Rousseau, G. W. Slater, Proceedings of the National Academy of Sciences USA, Vol. 84, pages 8011 to 8015 (1987).
Further U.S. Pat. Nos. selected as a result of a general computer search (LEXIS), some of which relate to gel electrophoresis, include 3,948,743; 4,059,501; 4,101,401; 4,181,501; 4,148,703; 4,207,166; 4,244,513; 4,322,225; 4,375,401; 4,391,688; 4,433,299; 4,541,910; 4,545,888; 4,552,640; 4,569,741; 4,608,146; 4,608,147; 4,617,103; 4,631,120; 4,632,743; 4,695,548; 4,707,233; 4,715,943; 4,729,823; 4,740,283; 4,747,918 and 4,794,075.
As a result of a patentability search there were selected U.S. Pat. Nos. 4,441,972; 4,473,452; 4,732,656; 4,737,251 and 4,786,387
U.S. Pat. No. 4,971,671, hereby incorporated by reference, illustrates the combination of known electrophoresis techniques and a new method of correlating the required field pulse characteristics and other process conditions with the size of the fragments to be resolved. Thus, in one embodiment a mixture of DNA particles is deposited in a conventional gel electrophoresis apparatus with a power supply and a single, uniform primary electric field having a positive voltage is applied in pulses in one direction. During the period between primary pulses, a secondary pulse of either a positive or a negative voltage is applied. Alternatively, during the period between primary pulses, secondary "pulses" of zero-field conditions may be applied. The aforementioned mixture of DNA fragments comprises in one embodiment a solution or gel sample containing DNA fragments of at least two different sizes.
For example, a mixture could contain fragments having 100,000; 200,000; 300,000; 400,000; and 500,000 base pairs. The durations of the primary and secondary pulses during the process are selected according to the formulae disclosed by G. W. Slater and J. Noolandi in "On the Reptation Theory of Gel Electrophoresis", Biopolymers, vol. 25, pages 431 to 454 (1986), hereby incorporated by reference.
Methods selected for separation of DNA fragments having more than 20,000 base pairs have several disadvantages. In some instances, except for the process as illustrated in the aforementioned '671 patent, commercially available electrophoresis equipment must be modified before these methods can be applied. For example, the process disclosed in the U.S. Pat. No. 4,473,452 patent pertaining to crossed gradient fields, requires extensive alterations to conventional gel electrophoresis apparatus. Also, many of the above described systems intended for separating DNA fragments of more than 20,000 base pairs, except for the process as illustrated in the '671 patent, use relatively high electric fields (above 3 volts/cm) necessitating the implementation of a bulky and expensive cooling system to avoid degradation of the gel and/or the DNA.i